The aim of this study was to evaluate the antimicrobial activity of bio-oil (obtained by pyrolysis of biomass: soybean oil, eucalyptus sawdust and coffee grounds) added to the blend B10 (diesel and biodiesel). The bio-oil is compatible to diesel oil and it contains promising compounds that can exhibit antimicrobial activity during fuel storage. We evaluated the antimicrobial activity of bio-oil added to diesel oil B10 by determining the minimum inhibitory concentration at different concentrations (0-10%) using oil-deteriorating microorganisms for 10 days at 30 °C. Bio-oil was separated from the aqueous phase by solid phase extraction using ultrapure water and sodium hydroxide. The aqueous phase containing the solubilized compounds of bio-oil was characterized by comprehensive two‑dimensional gas chromatography with time‑of‑flight mass spectrometric detection. Minimum inhibitory concentration in the range of 0.25% to 4% was observed for the tested inocula. The chromatographic analysis of both extracts allowed the identification of several oxygenated compounds, which the majority of the analytes consisted of phenolic compounds, followed by ketones.